Method of bacterial analysis



Feb. 2, 1960 E. J. POITRAS METHOD OF BACTERIAL ANALYSIS Original FiledNov. 22, 1954 INVENTOR. EDWARD J. POITRAS BY niuwkmmmwmimwnm ATTORNEYSMETHOD OF BACTERIAL ANALYSIS Edward J. Poitras, Holliston, Mass.,assignor to Millipore Filter Corporation, a corporation of MassachusettsOriginal application November 22,1954, Serial No; 470,169, now PatentNo. 2,879,207, dated March 24, 1959. Divided and this application April8, 1958, Serial No. 727,210

4 Claims. (Cl. 195-1035) The present invention comprises a novel method,of bacterial analysis whereby a sample to be analyzed may be filteredto collect the micro-organisms present, the cul* ture medium added, andthe organisms incubated, all Withidly and efficiently to determine theirsafety. It has been the practice in testing water to collect litersample s insealed containers, then to refrigerate them and transportthem to a laboratory where they are filtered and the bacteria arecultured. A recent improvement consists in utilizing small plastichermetically sealed dishes in con-' junction with portable filteringapparatus whereby filtration of the samples is carried out in the fieldand the filter membrane then placed and sealed in the dish. Con-;

veniently the dish is provided with a supporting pad for the filtersaturated'with a nutrient medium so that incubation may proceed duringthe time the sample is in transit to its final examination. Adisadvantage of this technique, however, is the possibility ofcontaminating the filter with extraneous organisms, particularly whenthe field work is under relatively septic conditions. Also, mostsuitable filter materials are quite fragile and delicate and theirtransfer from the filter apparatus to the dishes requires considerablecare, particularly if an underlying pad of nutrient medium is providedagainst which the filter must make complete face to face contact. 7

The bacterial analysis method-of this invention avoids the difficultiesencountered in previous techniques by utilizing a container adapted forboth filtration and incubation of microorganism specimens. The inventionthus eliminates all need for any manipulation of the filter,

avoids any danger of extraneous contamination, and obviates thenecessity of using separate filtering apparatus. In use a sample to beanalyzed is flowed through the unit, and the nutrient medium is thenintroduced and the unit sealed preparatory to incubation which iscarried out in the sealed unit.

In general, the method makes use of a bacterial anal-' nite Sttes PatenO 2,923,669 Patented Feb. 2, 1960 2 the top, and drained from the outletin the bottom. In passing through the unit the micro-organismsoriginally present in the sample are filtered out and collected on thefilter. They may then be cultured by introducing a culturemedium'through either the outlet port or inlet port to saturate theporous supporting pad and feed the overlying filter film. The inletand'outlet ports are then sealed and the unit is ready for incubation.

The filter film is preferably a microporous membrane suitable forremoving bacteria and other micro-organisms and retaining them on theouter surface. A satisfactory filter film is'described in US. Patent No.1,421,341, is-

' bodiments. Of these, preferred embodiments selected for ysis unitwhich comprises an enclosed container, conveniently in the form of aflat circular box, having a bottom and top each tapped to provide forthe introduction of the sample and removal of the filtrate throughsuitable connections. Against the bottom of the box is a poroussupporting pad, or other supporting structure providing drainage, onwhich rests a filter film held in place in face-to-face contact with thesupportingpad .by: an

annular member engaging the topsurface of the film at its marginaledges. When assembled, and prior to use, the inlet and outlet taps inthe bottom and top are preferably sealed, and the unit may then besterilizde and stored in a sterile condition until ready for use.

With a unit of this construction, the sample to be tested is passed, bysuitable connections, through the inlet in purposes of illustration aredescribed in detail below with reference to the accompanying drawings inwhich:

Fig. l'is a cross-sectional elevation of one embodiment of apparatususeful in this invention showing also appropriate connections forintroducing and removing samples to be tested;

Fig. 2 shows the parts of in exploded relation; v

Fig. 3 is a cross sec'tion'al elevation of another embodiment of thisinvention; and

the'embodiment in Fig. 1

Figs. 4 and 5 are schematic diagrams suggestive of preferred methodsofusing the bacterial analysis unit of this invention. v

In the embodiment shown in Fig. 1 and 2 the container 10 consists of abottom tray 12 centrally tapped to provide an outlet port 14, and aremovable close fitting close fitting cover 16 centrally tapped toprovide an inlet port 18. Conveniently, thebottom' tray 12 and cover 16are formed withside wall sections 2021 correspondingly tapered toprovide a rather rigid and close fit. "The outlet and inlet por'ts 14and 18 are preferably formed as inwardly tapering bores in outer collars22 and 24 which serve to facilitate the making of appropriateconnections to the ports and to strengthen the structure. Conveniently,the cover 16 and bottom tray 12 are formed of a transparent plastic,such as polystyrene with the top of the cover 16 smooth and flat topermit visual observation of the interior, and in general the preferredcontainer is constructed as described in US. Patent No.

The filter film 26 is supported on the bottom tray 12 by a poroussupporting pad 28, conveniently a circular piece of thick filter paperor felt'whic'h serves to provide drainage from the entire area beneaththe film 26 to the outlet port 14, and serves also as a bibulousstructure for absorbing nutrient medium for culturing micro-organismscollected on the filter film.

The filter film 26 isconveniently held in place byan underlyingresilient gasket ring 30, of for instance polychloroprene, surroundingthe pad 28 and supporting the lower marginal edge of the film, and acooperating fra'm'e 32 engaging the upper-marginal edgev of the film,

and extending upward into contact with the cover 16.

the lower marginal edges, of the filter film 26 are supported on araised shoulder; 40 formed as an integral part of the bottom tray 12,and the upper marginal edges are engaged by, a downwardly extendingflange 42 For field work it may be desired that the container be.

more positively sealed than by the friction fit of the bottom tray 12and cover 16, and for this purpose a band of pressure sensitive adhesivetape, eg a vinyl or cellulose tape, may be wrapped around the side walls20-21 I to prevent any possible opening and contamination of the unit.

In using the bacterial analysis unit of this invention to providebacterial cultures, of samples tested, connections to the inlet andoutlet ports 18 and 14 are conveniently made by tubular tapered fittings56v and 5,8; of polyethylene or other material suitable for insuring atight connection, inserted into the ports. A convenient technique fortaking Water samples, as suggested by Fig. 4, consists in connecting atube 57 through a fitting 56 to the inlet port collar 24 and immersingthe other end of the tube in the water to be tested. The outlet fitting58 is then connected to a source of vacuum, to draw the sample throughthe unit and then to drain the unit, after which the fittings areremoved, and a quantity of a culture medium necessary to saturate thesupporting pad is introduced, as by a syringe, through the outlet port14, the ports are then sealed preliminary to incubation as by plugs 51or pieces of pressure sensitive adhesive tape. A convenient means ofapplying the vacuum, particularly in field work, utilizes an evacuatedbottle 60 (Fig. 4) having a soft rubber stopper which is penetrated by ahollow needle 62.connected to the outlet fitting 58.

Alternatively, a sample may be filtered by placing it in a soft flexiblecontainer 64 of e.g. rubber or polyethylene, which is connected to theinlet port 18, and squeezing the container to force the sample throughthe unit, as suggested by Fig. 5. In this procedure, no connection tothe outlet port 14 is required.

After the sample of micro-organisms has been collected on the filterfilm, a conventional liquid nutrient medium may be introduced througheither the inlet or outlet ports 18 or 14 to saturate the pad 28, theinlet and outlet ports. are then sealed, as by a pressure sensitive tapeor a plug, and the unit is placed in incubation.

The unit of this invention is adapted to numerous bacteriologicalanalysis techniques. For instance, it may be used for the culture ofboth aerobic and anaerobic organisms. With aerobic organisms it is onlynecessary that the capacity of the container between the filter film 26and cover 16 be sufficient that the required amount of air will bepresent when the container is sealed. Where anaerobic organisms are tobe grown, the unit may be flushed with nitrogen or carbon dioxide toremove the air, and then sealed. The unit may also be usedadvantageously with techniques requiring two or more successive culturemedia, for instance, it is frequent practice to employ first anenrichment medium, and then a difierential medium, and this may be doneby draining off the enrichment medium after incubation thereon, as byopening the inlet and outlet ports and applying a vacuum to the latterwhile applying sterile air or other gas to the inlet, then introducingthe differential medium in the ordinary manner.

It will be understood that the foregoing description is by way ofillustration and that numerous modifications of the construction andutilization of the bacterial analysis unit readily occurring to thoseskilled in the art may be made without departing from the scope of thisinvention.

This application is a division of applicants co-pending applicationSerial No. 470,169 filed November 22, 1954, now Patent No. 2,879,207.

Having thus disclosed this invention and described in detail preferredembodiments thereof, I claim and desire to secure by Letters Patent:

1. The method of assaying for biological specimens comprising providingan enclosed container havinga drainage port and an inlet port and filterfilm adapted to remove micro-organisms mounted in said container on aporous supporting pad overlying the drainage port, introducing a fluidto be assayed into the inlet port and draining said fluid from theoutlet port to cause said fluid to flow through said filter film,thereby collecting biological specimens on the surface of said filterfilm, impregnating said supporting pad with a nutrient medium while saidfilter film is mounted thereon, closing said container, and incubatingthe specimens collected.

2. The method of assaying for biological specimens comprising providinga filter film adapted to remove micro-organisms mounted on a poroussupporting pad, flowing a fluid to be assayed through said filter filmto collect biological specimens on the unsupported surface thereof,impregnating said supporting pad with a nutrient medium while saidfilter film is mounted thereon, enclosing said pad and film in acontainer, and incubating the specimens collected.

3. The method of assaying for biological specimens comprising providingan enclosed container having a bottom tapped to provide a drainage portand a top tapped to provide an inlet port and a filter film adapted toremove micro-organisms mounted in said container on a porous supportingpad overlying the drainage port, introducing a fluid to be assayed intothe inlet port and draining said fluid from the outlet port to flow saidfluid through the filter film, thereby collecting biological specimenson said filter film, introducing a nutrient medium through one of saidports to impregnate said supporting pad therewith, closing the inletport and the outlet port, and incubating the specimens collected.

4. The method of assaying for biological specimens comprising providingan enclosed container having a bottom tapped to provide a drainage portand a top tapped to provide an inlet port and a filter film adapted toremove micro-organisms mounted in said container on a porous supportingpad overlying the drainage port, introducing a fluid to be assayed intothe inlet port and draining said fluid from the outlet port to fiow saidfluid through the filter film, thereby collecting biological specimenson said filter film, introducing a nutrient medium through said outletport to impregnate said supporting pad therewith, closing the inlet portand the outlet port, and incubating the specimens collected.

References Cited in the file of this patent UNITED STATES PATENTS GoetzMar. 16, 1954 Lovell May 5, 1954 OTHER REFERENCES

1. THE METHOD OF ASSYING FOR BIOLOGICAL SPECIMENS COMPRISING PROVIDINGAN ENCLOSED CONTAINER HAVING A DRAINAGE PORT AND AN INLET PORT ANDFILTER FILM ADAPTED TO REMOVE MICRO-ORGANISMS MOUNTED IN SAID CONTAINERON A POROUS SUPPORTING PAD OVERLYING THE DRAINAGE PORT, INTRODUCING AFLUID TO BE ASSAYED INTO THE INLET PORT AND DRAINAGE SAID DLUID FROM THEOUTLET PORT TO CAUSE SAID FLUID TO FLOW THROUGH SAID FILTER FILM, THERGYCOLLECTING BIOLOGICAL SPECIMENS ON THE SURFACE OF SAID FILTER FILM,IMPREGNATING SAID SUPPORTING PAD WITH A NUTRIENT MEDIUM WHILE SAIDFILTER FILM IS MOUNTED THEREON, CLOSING SAID CONTAINER, AND INCUBATINGTHE SPECIMENS COLLECTED.